Expression Of Envelope Protein VP28 Gene Of White Spot Syndrome Virus And Study On The Antigenicity Of Recombinant Protein

White. spot syndrome (WSS) is one of the most common and most disastrous diseases for shrimp worldwide. It causes up to 100% mortality within 7 to 10 days in commercial shrimp farms, resulting in large economic losses to the shrimp farming industry. As a result, WSSV resistant research has been given much attention in the present situation. In this study an envelope protein VP28 of WSSV was expressed both in E. coli and insect cells. Crayfish, Cambarus proclarkii, were intramuscularly injected with different recombinant VP28 and then challenged with WSSV to test the protective effects. This study was conducted to understand whether VP28 protein obtained from insect cells can bring better effect on crayfish survival upon WSSV challenge. The main research contents and results were as follows:1) Using crayfish as a proliferation system, a large amount of intact WSSV virion could be purified using 30% sucrose solution by a few steps of differential centrifugation. The morphology of the negatively stained purified WSSV virions was rod-shaped to somewhat elliptical with an integral envelope and each has a long tail-like appendage at one extremity. The size of the virus particles was about 80～120 nm in diameter and 360～420 nm in length and which of the nucleocapsid was about 80～90 nm in diameter and 320～400 nm in length. Using SDS-PAGE, at least 12 major and distant protein bands could be observed by Coomassie Brilliant R-250 staining.2) Envelope protein VP28 gene was amplified from WSSV genome. Nucleotide sequence analysis showed that VP28 was about 615 in size and it encoded postulated proteins of 204 amino acids. Its sequence was almost 99%～100% identical to that of other VP28 genes available in GenBank. Computer analysis of the 204 amino acids showed that protein sequences contain multiple putative N- and O-glycosylation sites.3) VP28 gene was expressed in E.coli and purified by using Ni-NTA affinity columns. The induced products were analyzed by SDS-PAGE and Western blot. Bands corresponding to the fusion proteins were observed at the expected heights. The objective band of recombinant VP28 was confirmed by Western analysis with anti-His6 monoclonal antibody. The size of the recombinant VP28 protein is smaller than its authentic counterpart protein from purified WSSV. This suggests that the VP28 gene product is made but not further processed in E.coli cells.4) Bac-to-Bac baculovirus expression system was used to express VP28 gene in order to gain properly folded proteins with biological activity. The VP28 fragment was cloned into a pFastBacTM donor plamid. The recombinant Bacmid-VP28 was formed after transposition. Insect cells Sf9 were transfected by the recombinant Bacmid DNA to express VP28. SDS-PAGE and Western blot analysis showed that the band corresponding to VP28 was observed at the expected height, indicating that VP28 was expressed in insect cells. The size of the recombinant VP28 protein is similar to that from purified WSSV but 6 kDa bigger than its theoretical size. This suggests that posttranslational modification of this protein may have been correctly performed in insect cells.5) In the preliminary trials to estimate the condition of WSSV infection, water temperature was proved to be one of the most important environmental factors affecting outbreaks of WSS. Mortality of crayfish increased as the water temperature rose from 18^C to 28℃, while hyperthermia could protect crayfish from WSS after challenge with WSSV. Competitive PCR showed that viral levels at 32±1℃remained at 10⁵ copies mg^-1 tissue while at 24±1℃levels were significantly higher, rising from 10⁴ to 10¹⁰ copies mg^-1 tissue. These results suggest that hyperthermia reduces viral replication but dose not eliminate viral particles from WSSV-infected crayfish.6) In order to investigate whether protein structure has an effect on antivirus of envelope protein of WSSV, crayfish were intramuscularly injected with the cell lysates infected with the recombinant pET-VP28 and AcNPV-VP28, respectively and then challenged by intramuscular injection of WSSV to assess the duration of protection. The crayfish injected with AcNPV-VP28 showed generally lower mortality rates compared to that injected with pET-VP28, resulting in RPS of 89% and 35% compared to the control groups, injected by wild AcNPV and pET-30a, respectively. This results show that anti-WSSV ability of VP28 protein obtained from insect cells is much better than that of VP28 protein obtained from E. coli. It might be inferred that correctly folded protein antigen could bring better immunology response in crayfish.